1. Technical Field
The present disclosure relates generally to multi-specific Fab fusion proteins (MSFP). In particular, the MSFP of the present disclosure comprise an antibody Fab fragment with both N-termini fused to a fusion moiety by a cleavable or non-cleavable linker. The Fab fragment of the MSFP specifically binds to native cell surface target antigens as well as certain soluble forms of the same antigen. One or both fusion moieties bind specifically to target antigens on cell surfaces.
2. Description of the Related Art
Conventional immunoglobulin G (IgG) is a tetrameric molecule comprising two identical immunoglobulin heavy chains and two identical immunoglobulin light chains. IgG heavy chain has a variable region at the N-terminus followed by the first constant region (CH1), a hinge and two additional constant regions (CH2CH3). IgG light chain is comprised of two domains: an N-terminal variable region and a C-terminal constant region. The heavy chain variable region (VH) interacts with the light chain variable region (VL) to constitute the minimal antigen binding region, Fv. The antigen binding region is stabilized by the interaction between the first constant region of heavy chain (CH1) and the light constant (CL) and further by the formation of a disulfide bond between the two constant regions (to form a Fab fragment). The homodimerization of CH2CH3 domains (to form Fc) and consequently the hinge disulfide bond formation stabilize the IgG structure. Thus an IgG has two antigen binding Fab arms which are relatively flexible in orientation with each other and with the Fc domain. In addition, the binding regions (which interact directly with antigen) are located at the N terminus with no further structures beyond. Conceivably, this structure feature facilitates antibody interaction with antigen with minimal interference from steric hindrance. This property is especially important for binding to cell surface antigen that is often located very close to the complex cell membrane.
In recent years, full length monoclonal antibodies have been successfully used to treat cancer, autoimmune and inflammatory diseases and other human diseases (Nelson, Nat. review, Drug Discovery (2010) 9:767-74). Although five different types of immunoglobulins (IgA, IgD, IgG, IgM and IgE) exist naturally, IgG represents the most suitable modality for human therapeutics because of the favorable properties including high binding affinity and specificity, high bioavailability, long serum half life in circulation, potential effector function capability and the industrial-scale manufacturability.
Monoclonal antibodies (non conjugated or naked antibody) currently approved by drug regulatory agencies worldwide for clinical use in oncology setting working generally by one or a combination of the following mechanisms: 1) blocking cell growth signaling, 2) blocking the blood supply to cancer cells, 3) directly mediating cell apoptosis, 4) eliciting immunological effector functions such as antibody dependent cellular cytoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and complement dependent cytotoxicity (CDC), and 5) promoting adaptive immunity towards tumors.
Monoclonal antibody therapies have demonstrated survival benefits in the clinic. However, the overall response rates in cancer patients are low, and the survival benefits are marginal (several months) compared to chemotherapy. Although the underlying reasons for the lack of robust clinical anti-cancer activities are not fully understood, research has suggested that cancer cells often quickly develop compensating signaling pathways to escape cell death. Also, cancer stem cells (CSC), which are considered as potent cancer initiating cells, are less active at cell proliferation therefore they tend to sustain the lack of growth signal better.
In recent years, ADCC was demonstrated to play a significant role in the clinical efficacy of anti cancer antibodies. Antibody Fc binds to Fc gamma receptors of which there are numerous forms: FcγRI, FcγRIIa, FcγRIIb FcγRIIc, FcγRIIIa and FcγRIIIb. The Fc domain has especially high affinity fo FcγRIIIa, which is expressed on natural killing (NK) cells, macrophages, and neutrophils. Binding of Fc to FcγRIIIa activates NK cells which can then destroy nearby cancer cells.
Engineered antibodies with enhanced activating FcγR binding properties via either protein engineering (involving a variety of amino acid mutations in the CH2 region) or production host cell (CHO) line engineering to reduce or eliminate fucosylation in the Asn297 glycan structure have been successfully tested in preclinical studies with improved biological activities. IgG antibodies containing enhanced effector functions are currently in clinical testing with the goal of improved efficacy. The current available clinical data indicate that these antibodies are very promising.
Another anti cancer therapeutic approach is to utilize T cells. T cells provide defense against cancer throughout life by patrolling the body in search for newly arisen cancer cells and eliminating them effectively and promptly. Successful therapeutic approaches harnessing T cell immunity in cancer treatment include: 1) the FDA approved use of PROLEUKIN® (aldesleukin, recombinant IL-2) for metastatic melanoma and metastatic kidney cancer; 2) FDA approved use of PROVENGE® (Sipuleucel-T) for asymptomatic metastatic hormone refractory prostate cancer. PROVENGE® is a dendritic cell vaccine that activates prostate cancer-specific cytotoxic T cells ex vivo, which cells are then reinfused into the patient; 3) FDA approve use of ipilimumab (anti CTLA-4 antibody to activate T cells by inhibiting T cell inhibitory signaling pathway) for advanced melanoma.
Because T cells do not express Fc gamma receptors (FcγR), anti tumor antibodies cannot effectively activate cytotoxic T cells directly. One of the many promising methods aimed to activate T cell for tumor killing purposes is to use bispecific antibodies (bsAb) to directly bring T cells to the proximity of tumor cells, resulting in activation of T cells and the killing the tumor cells. CD28 and CD137 (4-1BB) are two potent T cell co-stimulatory receptors utilized in bispecific targeting approaches. Examples include: CD28×NG2 (Grosse-Hovest et al., Eur J Immunol 33:1334-40, 2003), CD28×CD20 (Otz et al., Leukemia 23:71-7, 2009) and 4-1BB×CD20 (Liu et al., J Immunother 33:500-9, 2010). Other T cell surface targets capable of triggering T activation have also been used for retargeting them to tumor cells using bispecific antibodies. Various bispecific and multispecific antibody formats have been developed in the past and reviewed recently (Muller and Kontermann, Biodrugs 24: 89-98, 2010; Chames and Baty, mAbs 1:539-47, 2009; Deyev and Lebedenko, BioEssays 30:904-918, 2008). These formats fall into the following three large categories: 1) IgG-like bispecific molecules based on Fc heterodimerization or covalent fusion to the heavy or light chain, including quadroma technology (Staerz et al., PNAS 83:1453-7, 1986), knob and hole technology (Nat. Biotechnol. 16: 677-81; J. Biol. Chem. 285:19637-46, 2010), strand-exchange engineered domain “SEED” technology (Davis et al., PEDS 23:195-202, 2010), fusion to the C-terminus of IgG heavy or light chain (Coloma and Morrison, Nat. Biotechnol. 15:159-63, 1997; Shen et al., J. Immunol. Methods 318:65-74, 2007; orcutt et al., PEDS 23:221-8, 2010; Dong et al., J. Biol. Chem., 286:4703-17, 2011, Lazar et al., patent application, US20110054151), fusion to the N-terminus an IgG heavy or light chain (Shen, et al., J. Biol. Chem. 281:10706-17, 2006; Wu et al., Nat. Biotechnol. 25:1290-7, 2007), 2) Fc fusion bispecific antibodies (Mabry et al., PEDS 23:115-127, 2010; Miller et al., PEDS 23:549-557, 2010), 3) antibody variable region only molecules through fusion or nonconvalent association, including diabody (Db) (Holligger et al., PNAS 90:6444-8, 1993), disulfide bond linked diabody (also know as dual affinity re-targeting or DART) (Johnson et al., J. Mol. Biol. 399:436-49, 2010), single chain diabody (scpb) (Alt, et al., FEBS Letters 454:90-4, 1999), tandem diabody (tandAbs) (Kipriyanov, et al., J. Mol. Biol. 293:41-56, 1999), tandem single chain Fv (taFv) (Mack, et al., PNAS 92:7021-5, 1995), and 4) Fab based fusion molecules, including bibody and tribody (Schoonjans et al., J. Immunol. 165:7050-7, 2000; Website of Biotecnol SA, at the world-wide web address biotecnol.com), Fab fusion to single domain antibodies (patent application, US2010/0239582A1), and Fab′2-fusion (U.S. Pat. No. 5,959,083).
Efforts in the area of the bispecific antibody field over the last two decades started to bear fruits clinically. Cantomaxomab (REMOVAB®, an anti-CD3, anti-EpCAM trifunctional antibody), was approved in Europe for symptomatic malignant ascites in 2009. However, while bispecific antibodies have demonstrated potent tumor cell killing potential, severe side effects, including systemic immune activation, immunogenicity (anti drug antibody response) and general poor manufacturability of these molecules remain and to a large extent, have prevented this class of drugs from broad applications. Recently bispecific antibody technology platform referred to as bi-specific T cell engagers, or BiTEs, employing an anti-CD19 scFv-anti-CD3 scFv fusion (Blinatumomab), attracted a lot of attention because of its outstanding potency demonstrated in preclinical and clinical tests (Bargou et al., Science (2008) 321:974-7). In particular, patients with non-Hodgkin's lymphoma showed tumor regression, and in some cases complete remission during a clinical trial of blinatumomab administration. However, blinatumomab caused severe side effects including central nervous system side effects manifested by the loss of language ability and disorientation. Symptoms were transient and reversible once administration of the drug was stopped. It was hypothesized that the direct binding of the CD3 (on T cells) by the drug causing T partial activation and cell redistribution (patent application, US2010/0150918A1). Some of the redistributed T cells adhere to the brain miscro-vasculature, partially activate the endothelial cells and lead to the enhanced permeability of the micro-vasculature in the brain. It was observed in the clinic trial that the incidence of side effects was lower in patients with high B cell to T cell ratios than those with low ratios. It has also been reported that using different CD3 binding antibody fragments can alleviate or avoid T cell redistribution in Monkeys. These observations strongly suggest that antibody binding to CD3 together with the binding epitope on CD3 and the resultant partial activation of the T cell may be the direct cause of the severe CNS side effects.
Another drawback of the CD19×CD3 bispecific scFv-scFv fusion is that the drug requires daily intravenous infusion (i.v.) drug delivery due to its short half-life and incompatibility with subcutaneous administration. In addition, scFv-scFv fusion proteins have a tendency to aggregate. Therefore, BiTE molecules require highly complicated antibody engineering skills and it is laborious to make them stable and manufacturable.
Anti cancer activities of antibody drugs engaging FcγR or CD3-expressing immune cells demonstrated clinical proof of concept. However, the shortcomings of the current bispecific antibody formats remain a challenge for broad application of these drugs for treating cancer patients with good efficacy and safety profiles. Therefore, there remains an urgent need for new bispecific molecule designs with improved profiles on product efficacy, stability, safety and manufacturability.
References for further background information include: Coloma and Morrison, Nat. Biotechnol. 15:159-63, 1997; Kontermann, Acta Pharmacol. Sin., 26, 1-9. 2005; Marvin and Zhu; Acta Pharmacol. Sin., 26, 649-658.2005; Shen et al., J. Immunol. Methods, 318:65-74, 2007; Shen et al. JBC 281:10706-10714, 2006; Wu et al., Nat. Biotechnol., 25, 1290-1297, 2007; Orcutt Prot PEDS 23:221-8, 2010; Mabry PEDS vol. 23 no. 3 pp. 115-127, 2010; Schoonjans The Journal of Immunology, 2000, 165: 7050-7057; Michaelson, mAbs 1:2, 128-141; 2009; Robinson et al., British Journal of Cancer (2008) 99, 1415-1425.